The present invention relates to nucleic acid agents for downregulating H19 and use thereof for the treatment of cancer.
H19 was the first human imprinted non protein-coding gene to be identified showing expression of only the maternal allele. It is also imprinted in mice. H19 was mapped on the short arm of the human chromosome 11, band 15.5, homologous to a region of murine chromosome 7. It belongs to a group of genes that very likely does not code for a protein product. H19 gene is abundantly expressed in embryogenesis but is shut off in most tissues after birth. However, studies of various tumors have demonstrated a re-expression or an over-expression of the H19 gene when compared to healthy tissues. Moreover in cancers of different etiologies and lineages, aberrant expression in allelic pattern was observed in some cases. While H19 shows mono-allelic expression in most tissues throughout development, with the exception of germ cells at certain stages of maturation, and in extra-villous trophoblasts, bi-allelic expression of this gene, referred as “relaxation of imprinting” or “loss of imprinting” (LOI), have been found in an increasing number of cancers, for example, hepatocellular carcinoma, liver neoplasms of albumin SV40 T antigen-transgenic rats, lung adenocarcinoma, esophageal, ovarian, rhabdomyosarcoma, cervical, bladder, head and neck squamous cell carcinoma, colorectal, uterus and in testicular germ cell tumors. Today nearly 30 types of cancers show dysregulated expression of H19 gene as compared to healthy tissues, with or without LOI. For a recent review see Matouk et al (Matouk et al, 2005, Gene Ther Mol Biol).
It was also shown that H19 over-expression of ectopic origin conferred a proliferative advantage for breast epithelial cells in a soft agar assay and in several combined immunodeficient (SCID) mice (Lottin et al, 2002, Oncogene 21, 1625-1631). In tumors formed by the injection of cells of a choriocarcinoma-derived cell line (JEG-3), and a bladder carcinoma cell line (T24P), the H19 level is very high when compared to the level of H19 in cells prior to injection [Rachmilewitz et al, 1995, Oncogene 11, 863-870].
Moreover, certain known carcinogens upregulate the expression of the H19 gene. A dramatic elevation of H19 RNA levels was detected in the airway epithelium of smokers without (LOI) [Kaplan et al, 2003, Cancer Res 63, 1475-1482]. BBN (a known carcinogen of the bladder) also induces the expression of H19 gene in the rat model of bladder cancer [Ariel et al, 2004, Mol Carcinog 41, 69-76]. Likewise, Diethylnitrosamine (a known carcinogen of the liver) induces the expression of H19 in a mice model of hepatocellular carcinoma [Graveel et al, 2001, Oncogene 20, 2704-2712]. All of these observations and others contradict the initial proposal that H19 is a tumor suppressor gene.
Comparing patterns of gene expression in two homogeneous cell populations that only differ in the presence or absence of H19 RNA have identified plenty of downstream effectors of H19 RNA, among these are group of genes that were previously reported to play crucial roles in some aspects of the tumorigenic process. H19 RNA presence may enhance the invasive, migratory and angiogenic capacity of the cell by up regulating genes that function in those pathways, and could thus contribute at least to the initial steps of the metastatic cascade. Additional studies highlight H19's potential role in promoting cancer progression and tumor metastasis by being a responsive gene to HGF/SF.
The specific expression of H19 gene in cancer cells has prompted its use in clinical applications for diagnosing cancer.
Thus, U.S. Pat. No. 5,955,273 to the present inventors teaches the use of H19 gene as a tumor specific marker.
PCT Pub. No. WO 9524503 teaches the detection of malignancies and their grading with a H19 gene probe by in-situ hybridization—useful for detecting presence/absence of malignancy in pediatric Wilms' Tumor.
PCT Pub. No. WO 0403159 teaches down-regulation of H19 for treating diseases associated with angiogenesis, such as cancer. However, down-regulation of H19 was not shown to reduce tumor size or volume, neither was specific and efficacious siRNA agents capable of down-regulating H19 taught. Furthermore, PCT Pub. No. WO 0403159 does not teach use of anti H19 agents as part of a combination therapy for treating cancer.
There is thus a widely recognized need for, and it would be highly advantageous to have, methods and compositions for down-regulating H19 for cancer treatment.